The University has an internationally recognized snake venom research program and has a serpentarium with 150 poisonous snakes from 16 different species. The proposed research will produce scientifically and medically important knowledge about metalloproteinase specificity which have important physiological and pathological functions Venoms are abundant sources of stable metalloproteinases that can be isolated in large quantities sufficient for use in crystallographic studies. Many mammalian metalloproteinases are found in low concentrations and difficult to obtain. Venom metalloproteinases share a high degree of homology with mammalian metalloproteinases such as type IV collagenase are involved in extracellular matrix digestion, which is a critical component of many physiological (e.g., wound healing, tissue remodeling, and differentiation) and pathological (e.g., tumor cell invasion, metastasis, and arthritis) conditions. Similar active sites between mammalian metalloproteinases and the very stable venom metalloproteinases make it possible to use venom enzymes as models for the design of useful pharmaceuticals. This problem will be approached by first designing a fluorogenic substrate for snake metalloproteinases based on the cleavage site sequence from the oxidized insulin B chain. Flurogenic substrates can be prepared by using homologous substitutions to introduce tryptophan and p-nitrorphenylalanine into a peptide substrate. Fluorogenic substrates prepared in this way are useful tools for investigating proteinase mechanisms. The initial substrate specificity of snake metalloproteinases will be determined by the cleavage of B chain of insulin. Mass spectrometry will be used to determine the cleavage site. Other procedures could be used, but mass spectrometry analysis is the most sensitive, reliable, and elegant procedure. From preliminary information of the preferred cleavage site sequence, several analogs will be synthesized. The efficiency of the inhibitors will be determined by measuring the enzyme specificity constant (Kcat/Km). With these peptides, proteinase activity can be easily assayed in a continuous manner. It will be determined if these protease inhibitors block hemorrhage. Additionally, cleavage products will be used from basement membrane proteins such as collagen type IV to design metalloproteinase inhibitors. The long term goals are to determine if metalloproteinase inhibitors can neutralize hemorrhagins in snake venom as well as metalloproteinases in other pathological processes.